Barcode Crosstalk in ONT Multiplex Sequencing: Quantification and Mitigation Strategies

Multiplexing samples in long-read sequencing with Oxford Nanopore Next Generation Sequencing Technology (ONT) by ligating specific native barcodes to individual DNA samples enables significant increases of high throughput sequencing combined with a significant reduction of sequencing costs. However, this advantage carries the risk of barcode misassignment / crosstalk. Employing ONT multiplex sequencing with samples, we observed misassigned barcodes so called barcode crosstalk, after ONT library preparation according to the standard protocol, particularly in samples with low input DNA concentrations. We assumed that these barcode misassignments are largely due to misligation of remaining native barcodes during subsequent the subsequent sequencing adapter ligation. To systematically investigate and quantify barcode crosstalk, genomic DNA (gDNA) from four bacterial type strains with different DNA input concentrations was prepared using three protocols for library preparation: the Nanopore standard protocol (protocol A: version valid until July 2, 2025) the new Nanopore protocol (protocol B: version from July 2, 2025), and an in house protocol with pooling of the barcoded samples only after the sequencing adapter ligation step (protocol C: in house). All samples were sequenced on a Nanopore PromethIon device. The results clearly showed that the use of protocol A resulted in a pronounced barcode crosstalk especially detectable in samples with low DNA input concentrations (up to 2.4% misassigned reads). The ONT adjustment in protocol B (altered washing buffer vs. protocol A) significantly alleviated the barcode crosstalk to below 0.01%, whereas protocol C eliminated barcode crosstalk virtually completely. These observations emphasize that sequencing results obtained with older ONT native barcoding protocol variants should be critically reviewed. The newer ONT barcoding protocol is preferable for sequencing, but it does not completely eliminate the barcode crosstalk effect. In conclusion, for low DNA input and high accuracy sequencing, protocol C is recommended.